Filtration system and use thereof

ABSTRACT

The invention relates to a filtration system for use in a method of determining the presence and/or amount of cells, for example, viable cells, in a liquid sample, and to methods of using and manufacturing such a filtration system. The filtration system includes a cup with an upper portion and a ring portion, where the ring portion is separably coupled to the upper portion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/858,495, filed Sep. 18, 2015, which is a continuation ofInternational Patent Application No. PCT/US2014/063950, filed Nov. 4,2014, which claims priority to and the benefit of U.S. ProvisionalPatent Application No. 61/899,436, filed Nov. 4, 2013, the entiredisclosures of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to a filtration system for harvestingcells, for example, viable cells, in a liquid sample for later analysis,and to methods of using and manufacturing such a filtration system.

BACKGROUND

Microbial contamination by, for example, Gram positive bacteria, Gramnegative bacteria, and fungi, for example, yeasts and molds, may causesevere illness and, in some cases, even death in human and animalsubjects. Manufacturers in certain industries, for example, food, water,cosmetic, pharmaceutical, and medical device industries, must meetexacting standards to verify that their products do not contain levelsof microbial contaminants that would otherwise compromise the health ofa consumer or recipient. These industries require frequent, accurate,and sensitive testing for the presence of microbial contaminants to meetcertain standards, for example, standards imposed by the United StatesFood and Drug Administration or Environmental Protection Agency.

Depending upon the situation, the ability to distinguish between viableand non-viable cells can also be important. For example, during themanufacture of pharmaceuticals and biologics, it is important that thewater used in the manufacturing process is sterile and free ofcontaminants. Furthermore, it is important that water contained inmedicines (for example, liquid pharmaceutical and biological dosageforms, for example, injectable dosage forms) and liquids (for example,saline) that are administered to a subject, for example, vianon-parenteral routes, is also sterile and free of contaminants. On theother hand, the presence of some viable microorganisms in drinking watermay be acceptable up to a point. In order to be potable, drinking watermust meet exacting standards. Even though microorganisms may be presentin the water supply, the water may still be acceptable for humanconsumption. However, once the cell count exceeds a threshold level, thewater may no longer be considered safe for human consumption.Furthermore, the presence of certain predetermined levels ofmicroorganisms in certain food products (for example, fresh produce) anddrinks (for example, milk) may be acceptable. However, once those levelshave been exceeded the food or drink may be considered to have spoiledand no longer be safe for human consumption.

Traditional cell culture methods for assessing the presence of microbialcontamination and/or the extent of microbial contamination can takeseveral days to perform, which can depend upon the organisms that arebeing tested for. During this period, the products in question (forexample, the food, drink, or medical products) may be quarantined untilthe results are available and the product can be released. As a result,there is a need for systems and methods for rapidly detecting (forexample, within hours or less) the presence and/or amount of microbialcontaminants, in particular, viable microbial contaminants, in a sample.An important part of the process is capturing the cells to be analyzed,which must be completed in a quick, safe and consistent manner to enablethe efficiency of the overall detection system and methods.

SUMMARY

The invention is based, in part, upon the discovery of an improvedfiltration system (also referred to herein as a cell capture system)that can be used with a cell detection system to determine the presenceof viable cells in a cell containing sample. The filtration system canbe used in combination with an optical detection system that detects thepresence of cells, for example, viable cells in the sample. The resultscan be used to measure the bioburden (for example, to measure the numberand/or percentage and/or fraction of viable cells) of a particularsample of interest.

The filtration system described herein provides a number of advantagesover existing filtration systems. For example, the filtration systemminimizes the risk of leakage of fluid sample around the membrane thatcaptures cells thereby reducing the risk of inadvertently contaminatingthe portion of the filtration system that is touched or handled by theuser or that is placed within a cell detection system. Furthermore, thefiltration system is more user friendly than existing filtration systemsas it requires a fewer manipulation steps during operation by a user,which can be advantageous given that each additional manipulation stephas the potential to introduce contaminants into the sample beinganalyzed or the fluid sample being analyzed may inadvertentlycontaminate the user or the surrounding environment. This minimizes therisk of contaminating the user, the detection system, or the surroundingenvironment.

In one aspect, the invention provides a filtration system (cell capturesystem) for receiving a fluid sample. The system includes a cup havingan upper portion and a ring with a periphery. The upper portion isseparably coupled to the ring. The cup also includes a fluid permeablemembrane attached to the periphery to produce a fluidic seal between themembrane and the ring. A portion of the membrane is adapted to retaincells thereon. The system also includes a base configured to receive thering.

The membrane portion can (i) define a plurality of pores having anaverage diameter less than about 1 μm so as to permit fluid to traversethe second portion of the membrane while retaining cells thereon and(ii) be substantially non-autofluorescent when exposed to light having awavelength in a range from about 350 nm to about 1000 nm. Moreover, themembrane portion may have a flatness tolerance of up to about 100 μm(i.e., within ±50 μm). In some embodiments, the cup is adapted to directa fluid, when introduced into the upper portion, toward the membraneportion.

In certain embodiments, the ring is integrally formed with the upperportion of the cup. The ring can be separably coupled to the upperportion with a frangible connection, which can be a thin wall at anintersection of the upper portion and the ring. The frangible connection(e.g., a thin wall intersection) may define a circumferential groove.The frangible connection may also define a parting plane between theupper portion and the ring upon the application of a force sufficient tobreak the frangible connection. In other embodiments, the ring isseparably coupled to the upper portion via at least one of a threadedconnection, a bayonet connection, and an interference fit.

In some embodiments, the ring has a circumferential registrationfeature. The ring can include a plurality of protrusions about theperiphery of the ring, and one of the plurality of protrusions has atleast one of a width, a height, a thickness, and a spacing differentthan each of the other protrusions, which can act as a spatial register.In certain embodiments, the membrane is at least one of adhered, bonded,heat welded, and ultrasonically welded to the ring.

The base may include a cylindrical wall for receiving the ring. Incertain embodiments, the cylindrical wall defines a plurality of notchesthat mate with a plurality of protrusions of the ring. One of thenotches can be adapted to receive one of the plurality of protrusionshaving at least one of a width, a height, a thickness, and a spacingdifferent than each of the other protrusions, which can facilitatespatial registration. The cylindrical wall can define a circumferentialopening adapted to provide access to the ring, when the ring is disposedwithin the base, and can also define a recess adapted to receive amembrane support. The recess may define a plurality of openings adaptedto permit the passage of fluid therethrough. In one embodiment, the baseincludes a registration feature, which may include a depression definedby a surface of the base.

The cup may further include at least one latch adapted to couple the cupto the base to provide the appropriate frictional interfit between thecup and the base so as to permit the appropriate level of torque tofacilitate separation of the ring (with the associated membrane) fromthe upper portion of the cup but yet also permit subsequent removal ofthe ring (with the associated membrane) from the base. The latch may beadapted to resist separation of the cup and the base in a planeperpendicular to a parting plane, but not resist rotation of the cuprelative to the base.

In another aspect, the invention provides a method of harvesting cells,if present in a fluid sample. The method includes introducing the fluidsample to the upper portion of the cup described above and permittingthe fluid to pass through the membrane portion. In some embodiments, themethod includes, after applying the fluid, separating the upper portionfrom the ring. Separating the upper portion from the ring can includeapplying a force sufficient to decouple the ring from the upper portion,which force may be applied by twisting the cup relative to the base. Themethod may also include, after applying the fluid, securing a plug to abottom of the base.

In still another aspect, the invention provides a method ofmanufacturing the cell capture system described above. The methodincludes providing a ring with a periphery, securing a fluid permeablemember to a periphery to produce a fluidic seal between the membrane andthe ring, and positioning the ring having the membrane secured theretowithin a base configured to receive the ring. In some embodiments, themethod includes, prior to producing the fluidic seal, separably couplingthe ring to the upper portion. The positioning step may include matingthe cup with the base in a predetermined circumferential orientation. Inone embodiment, prior to positioning the cup within the base, the methodincludes placing a porous support in a recess formed in the base.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

FIGS. 1A and 1B are schematic top and bottom perspective views,respectively, of an exemplary cell capture system;

FIGS. 1C, 1D, and 1E are schematic side, top and bottom views of theexemplary cell capture system of FIGS. 1A and 1B;

FIG. 1F is a schematic cross-section view of the exemplary cell capturesystem taken along line F-F of FIG. 1D;

FIGS. 2A and 2B are schematic top and bottom perspective views,respectively, of an exemplary cup, which is part of the exemplary cellcapture system of FIGS. 1A-1F;

FIGS. 2C, 2D, and 2E are schematic side, top and bottom views of theexemplary cup of FIGS. 2A and 2B;

FIG. 2F is a schematic cross-section view of the exemplary cup takenalong line F-F of FIG. 2D;

FIGS. 3A and 3B are schematic top and bottom perspective views,respectively, of an exemplary base, which is part of the exemplary cellcapture system of FIGS. 1A-1F;

FIGS. 3C, 3D, and 3E are schematic side, top and bottom views of theexemplary base of FIGS. 3A and 3B;

FIG. 3F is a schematic cross-section view of the exemplary base takenalong line F-F of FIG. 3D;

FIGS. 4A and 4B are schematic top and bottom perspective views,respectively, of an exemplary base assembly including the exemplary baseof FIGS. 3A-3F;

FIGS. 4C, 4D, and 4E are schematic side, top and bottom views of theexemplary base assembly of FIGS. 4A and 4B;

FIG. 4F is a schematic cross-section view of the exemplary base assemblytaken along line F-F of FIG. 4D;

FIG. 5A is a schematic top perspective view of an exemplary cell capturesystem;

FIGS. 5B-5D are schematic side, top and bottom views, respectively, ofthe exemplary cell capture system of FIG. 5A;

FIG. 5E is a schematic cross-section view of the exemplary cell capturesystem taken along line D-D of FIG. 1D;

FIG. 5F is a schematic bottom perspective view of the exemplary cellcapture system of FIG. 5A;

FIG. 6A is a schematic top perspective view of an exemplary cup, whichis part of the exemplary cell capture system of FIGS. 5A-5F;

FIGS. 6B-6D are schematic side, top and bottom views, respectively, ofthe exemplary cup of FIG. 6A;

FIG. 6E is a schematic cross-section view of the exemplary cup takenalong line D-D of FIG. 6D;

FIG. 6F is a schematic bottom perspective view of the exemplary cup ofFIG. 6A;

FIG. 7A is a schematic top perspective view of an exemplary base, whichis part of the exemplary cell capture system of FIGS. 5A-5F;

FIGS. 7B-7D are schematic side, top and bottom views, respectively, ofthe exemplary base of FIG. 7A;

FIG. 7E is a schematic cross-section view of the exemplary base takenalong line C-C of FIG. 7C;

FIG. 7F is a schematic bottom perspective view of the exemplary base ofFIG. 7A;

FIG. 8A is a schematic top perspective view of an exemplary adapter foruse with a ring of a cup having a membrane;

FIGS. 8B-8D are schematic side, top and bottom views, respectively, ofthe exemplary adaptor of FIG. 8A;

FIG. 8E is a schematic bottom perspective view of the exemplary adaptorof FIG. 8A.

FIG. 9A is a schematic bottom perspective view of an exemplary lens foruse with the exemplary adapter of FIG. 8A;

FIGS. 9B-9D are schematic side, bottom, and top views, respectively, ofthe exemplary lens of FIG. 9A; and

FIG. 9E is a schematic top perspective view of the exemplary lens ofFIG. 9A.

DESCRIPTION

The instant invention is directed to a filtration system (cell capturesystem), a method of capturing/harvesting cells (including viable cells)in the filtration system, and a method for manufacturing the filtrationsystem. The cell capture system and related methods can be used, eitheralone or in combination, to capture/harvest cells for later analysis,e.g., to determine the bioburden (e.g., to measure the number and/orpercentage and/or fraction of viable cells in a sample) of a particularsample of interest.

The cell capture system can be used to capture cells (e.g., microbialcells, e.g., bacterial, yeast, or fungal cells) from a variety ofsources, including from a liquid sample (e.g., a water sample), acomestible fluid (e.g., wine, beer, milk, baby formula or the like), abody fluid (e.g., blood, lymph, urine, cerebrospinal fluid or the like),growth media, a liquid sample produced by harvesting cells from a sourceof interest (e.g., via a swab) and then dispersing and/or suspending theharvested cells, and a liquid (e.g., buffer or growth media).

Each of the various aspects and certain embodiments of the inventionwill be discussed in detail below.

(I) Cell Capture System

The cell capture system described herein can be used with an opticaldetection system that detects the presence of viable cells. The resultscan be used to measure the bioburden (e.g., to measure the number and/orpercentage and/or fraction of viable cells in a sample) of a particularsample of interest. Exemplary detection systems are described, forexample, in International Patent Application No. PCT/IB2010/054965,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,402, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054966,filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,380, filedFeb. 24, 2011, International Patent Application No. PCT/IB2010/054967,filed Nov. 3, 2010, and U.S. patent application Ser. No. 13/034,515,filed Feb. 24, 2011.

The cell capture system described herein provides a number of advantagesover existing cell capture systems. For example, the cell capture systemdescribed herein minimizes the risk of leakage of fluid sample aroundthe membrane and, as a result, the fluid being characterized must passthrough the membrane. This reduces the risk of inadvertentlycontaminating the portion of the cell capture system that is touched orhandled by the user or that is placed within the detection system.Furthermore, the cell capture system described herein is more userfriendly than other systems as it reduces the number of subsequentmanipulation steps during operation by a user, which can be advantageousgiven that each additional manipulation step has the potential tointroduce contaminants into the sample being analyzed or the fluidsample being analyzed may inadvertently contaminate the user or thesurrounding environment. This minimizes contamination of the user ordetection system or the surrounding environment. Furthermore, becausethe membrane is attached to a periphery of the ring, the membrane issubstantially planar and can be created to have a flatness within adesired flatness tolerance necessary to keep the membrane within thefocal plane of the detection system of certain optical detectors duringoperation.

One embodiment of a cell capture system 100, as shown schematically inFIGS. 1A-1F, includes a cup 105 and a base 110. A lid 115 to cover thecup 105 is optionally provided. The lid 115 can protect the inside ofthe cup 105 before use, such as during transport or preparation of afluid to be applied. The cell capture system 100 may be shipped in theassembled, ready-to-use state, for example, as depicted in FIGS. 1A-1F,allowing a user to quickly capture or harvest cells without additionalassembly, as described in greater detail below. The individualcomponents of the cell capture system 100 are described in greaterdetail below.

The cup 105 comprises an upper portion 230, a ring 235, and a fluidpermeable membrane 240, as depicted in FIGS. 1F and 2A-2F. The upperportion 230 is generally configured to direct fluid applied thereintoward a lower portion of the cup 105 where the ring 235 and themembrane 240 are disposed. The ring 235 is separably coupled to theupper portion 230 so that, when desired, the ring 235 and the membrane240 are retained while the upper portion 230 is disposed of separately.The ring 235 and associated membrane 240 can then be transferred to theoptical detection system for analysis of the membrane.

In some embodiments, the upper portion 230 and the ring 235 areintegrally formed together (e.g., through molding), with separabilityenabled through a frangible connection 245. The frangible connection 245can take many forms, including a locally thinned wall section (e.g., athickness of about 0.25 mm (0.0098 in.) instead of 1.5 mm (0.059 in.)for other parts of the cup 105) at an intersection of the upper portion230 and the ring 235. The thinned wall section may resemble a groove atthis intersection, and the frangible connection 245 may form a grooveeven if the wall section is not locally thinner. The frangibleconnection 245 defines a parting plane 250 (see, FIG. 2C), along whichthe upper portion 230 and the ring 235 are broken apart upon theapplication of a sufficient force (e.g., a torque of approximately 5-20inch pounds (0.56-2.26 newton meters) or more). Grips 252 may beprovided on the cup 105 and/or the base 110 to help the user apply thenecessary force without slippage. The grips 252 can be a variety offorms, such as the depicted strips, or another surface feature whichwould increase grip for the user, such as raised spots. In otherembodiments, the upper portion 230 and the ring 235 are formedseparately, then later joined together through another connection, suchas a threaded connection, a bayonet connection, and/or an interferencefit.

In the depicted embodiment (see, FIG. 2F), the membrane 240 isconnected, e.g., via heat welding or ultrasonic welding, to the ring 235about a periphery of the ring that surrounds an opening 255. Themembrane 240 may be otherwise adhered or bonded to the periphery of thering 235, such as with a mechanical fastener or an adhesive. Theconnection may or may not be permanent. It is desirable to create afluidic seal to restrict any fluid within the cup 105 from exiting orevacuating from the cup other than through the membrane 240. The planarmembrane 240 has an exposed first surface (i.e., the side facing theinterior of the ring 235), at least a portion of which is adapted toretain cells thereon. The membrane portion can: (i) define a pluralityof pores having an average diameter less than about 1 μm so as to permitfluid to traverse the portion of the membrane while retaining cellsthereon; and/or (ii) be substantially non auto-fluorescent when exposedto light having a wavelength in a range from about 350 nm to about 1000nm. Optionally, the membrane portion also can have a flatness toleranceof up to about 100 μm. A flatness tolerance specifies a tolerance zonedefined by two parallel planes within which the surface must lie. Forexample, in the embodiment described above having a flatness toleranceof up to about 100 μm, every point on the portion of the membrane 240falls between two parallel planes spaced 100 μm apart.

The membrane 240 can be any of a variety of shapes, e.g., circular,annular, ovoid, square, rectangular, elliptical, etc., and can have someportion or all of one side exposed for cell retention. In oneembodiment, the membrane 240 may be in the shape of a disc, e.g., asubstantially planar disc. In certain embodiments, the portion of theporous membrane 240 for capturing cells and/or particles is greater than400 mm², 500 mm², 600 mm², 700 mm², 800 mm², 900 mm² or 1,000 mm². Incertain embodiments, the portion of the porous membrane 240 forcapturing cells and/or particles is greater than 0.5 cm², for example,from 0.5 cm² to 300 cm², from 0.5 cm² to 100 cm², from 0.5 cm² to 50cm², from 1 cm² to 300 cm², from 1 cm² to 100 cm², from 1 cm² to 50 cm²,from 5 cm² to 300 cm², from 5 cm² to 100 cm², from 5 cm² to 50 cm². Themembrane 240 (e.g., in the form of a disc) can have a thickness in arange selected from the group consisting of approximately from 1 μm to3,000 μm, from 10 μm to 2,000 μm, and from 100 μm to 1,000 μm. Incertain embodiments, the membrane may have a thickness of about 0.020″.

The porous membrane 240 defines a plurality of pores having an averagediameter less than about 1 μm so as to permit fluid to traverse themembrane 240 while retaining cells thereon. In certain embodiments, theaverage pore diameter is about or less than about 0.9 μm, 0.8 μm, 0.7μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or 0.05 μm. Incertain embodiments, the average pore diameter is about 0.2 μm, and inother embodiments the average pore diameter is about 0.4 μm. Suitablemembranes 240 can be fabricated from nylon, nitrocellulose,polycarbonate, polyacrylic acid, poly(methyl methacrylate) (PMMA),polyester, polysulfone, polytetrafluoroethylene (PTFE), polyethylene andaluminum oxide.

In addition, the porous membrane 240 is substantiallynon-autofluorescent when exposed to light having a wavelength in therange from about 350 nm to about 1,000 nm. As used herein with referenceto the porous membrane 240, the term “substantially non-autofluorescentwhen exposed to a beam of light having a wavelength in the range fromabout 350 nm to about 1,000 nm” is understood to mean that the porousmembrane 240 emits less fluorescence than a fluorescently labeled cellor a fluorescent particle disposed thereon when illuminated with a beamof light having a wavelength, fluence and irradiance sufficient to causea fluorescence emission from the cell or particle. It is understood thata user and/or detector should be able to readily and reliablydistinguish a fluorescent event resulting from a fluorescent particle ora fluorescently labeled cell from background fluorescence emanating fromthe porous membrane 240. The porous membrane 240 is chosen so that it ispossible to detect or visualize a fluorescent particle or afluorescently labeled cell disposed on such a porous membrane 240. Incertain embodiments, the fluorescence emitted from a region of theporous membrane 240 (e.g., a region having approximately the samesurface area as a cell or cell colony or particle being visualized)illuminated with a beam of light may be no greater than approximately30% (e.g., less than 30%, less than 27.5%, less than 25%, less than22.5%, less than 20%, less than 17.5%, less than 15%, less than 12.5%,less than 10%, less than 7.5%, less than 5%, or less than 2.5%) of thefluorescence emitted from a fluorescent particle or a fluorescentlylabeled cell, when measured under the same conditions, for example,using a beam of light with the same wavelength, fluence and/orirradiance.

Suitable membranes 240 that are non-autofluorescent can be fabricatedfrom a membrane, e.g., a nylon, nitrocellulose, polycarbonate,polyacrylic acid, poly(methyl methacrylate) (PMMA), polyester,polysulfone, polytetrafluoroethylene (PTFE), or polyethylene membraneimpregnated with carbon black or sputtered with an inert metal such asbut not limited to gold, tin or titanium. Membranes 240 that have theappropriate pore size which are substantially non-autofluorescentinclude, for example, ISOPORE™ membranes (Merck Millipore), NUCLEOPORE™Track-Etched membranes (Whatman), ipBLACK Track Etched Membranes(distributed by AR Brown, Pittsburgh, Pa.), and Polycarbonate (PCTE)membrane (Sterlitech).

In certain embodiments, the ring 235 has a circumferential registrationfeature to ensure proper positioning of the ring 235 in the base 110,and which also provides a consistent orientation for the membrane 240.Having a consistent orientation allows for reference to specificlocations of cells on the membrane 240 (for example, the viable cells)retained on at least a portion of the membrane 240. For a disc shapedmembrane, polar coordinates (i.e., radial “r” and angular “θ” coordinatelocations) may be suitable. In some embodiments, the registrationfeature includes a plurality of protrusions 260 (see, FIGS. 2A and 2C),for example, at least two, three, four, five, six, seven, eight, nine,or ten protrusions spaced about the periphery of the ring 235. In oneembodiment, the ring defines at least four protrusions. To properlyorient the ring 235, and thereby the membrane 240, one of theprotrusions 260′ may be different than the other protrusions, such ashaving a different width, height, thickness, and/or spacing (e.g.,spaced 60 degrees from another protrusion 260 instead of 90 degrees). Acorresponding feature on the base (see below for more detail) issimilarly sized to receive the protrusions 260, including the uniqueprotrusion 260′, so that the ring 235 and the base 110 may only becoupled in one orientation. The protrusions 260 can also be provided ina substantially symmetrical layout such that multiple orientations arepossible.

The base 110, depicted in FIGS. 3A-3F, has a cylindrical wall 265 forreceiving the ring 235. While cylindrical in the depicted embodiment,the wall 265 may be a variety of shapes complementary to the shape ofthe ring 235, such that the ring 235 could be received within the wall265. The wall 265 defines a plurality of notches 270 sized and adaptedto mate with the protrusions 260. A unique notch 270′ can be sized orpositioned to receive the unique protrusion 260′, thereby achieving aconsistent frame of reference. The protrusions 260 and the notches 270are also dimensioned to provide sufficient frictional interfit so asmaintain engagement when an appropriate torque is applied to ring235/base 110 relative to the upper portion of the cup 230 at which pointring 235 breaks away from the upper portion of the cup 230 along partingplane 250. An overlap of approximately 0.787 mm (0.030 in.) may besufficient, although greater, and lesser, overlaps can also work. Thewall 265 also defines a circumferential opening 275 providing access tothe ring 235 when it is disposed within the base 110 (as depicted inFIGS. 3A, 3D, 4A, and 4F). For example, depending upon thecircumstances, a user can remove ring 235 together with membrane 240 byinserting an instrument, for example, forceps, through the opening 275,to remove the ring.

The base 110 further defines a recess 280 (see, FIG. 3F) for receiving afluid permeable membrane support 285, that is optionally substantiallyplanar. The membrane support 285 is permeable and adapted to contact abottom surface of the membrane 240 when the ring 235 is disposed in thebase 110. The fluid permeable support 285, for example, in the form of asmooth planar porous plastic frit, retains enough fluid to maintainmoisture in the porous membrane 240 disposed adjacent the permeablesupport 285, which in certain embodiments, can be important to maintainthe viability of cells retained on the porous membrane 240. The support285 can have a thickness in a range selected from the group consistingof approximately from 0.1 mm to 10 mm, from 0.5 mm to 5 mm, and from 1mm to 3 mm. The recess 280 defines openings 290 to permit the passage offluid therethrough.

In order to facilitate accurate detection and count estimation of thecaptured cells, it is beneficial (even essential in some instances,depending on the configuration and capabilities of the detection system)that the membrane 240 is substantially planar (e.g., substantiallywrinkle free) during cell detection. As used herein, the term“substantially planar” is understood to mean that an article has aflatness tolerance of less than approximately 100 μm (i.e., within +50μm). This is because height imperfections (e.g., wrinkles) may interferewith the optical detection/measurement system, leading to erroneousresults. As a result, it can be important for the porous membrane 240when dry and/or wet and depending on detection conditions), retains arelatively tight flatness tolerance, within the detection capability ofthe detection system.

Under certain circumstances, depending upon the detection systememployed, the membrane (and the cells disposed therein) is maintainedwithin a tight flatness tolerance (e.g., within a flatness tolerance ofup to about 100 μm (±50 μm), e.g., up to about 10 μm (±5 μm), up toabout 20 μm (±10 μm), up to about 30 μm (±15 μm), up to about 40 μm (+20μm), up to about 50 μm (±25 μm), up to about 60 μm (±30 μm), up to about70 μm (±35 μm), up to about 80 μm (±40 μm), up to about 90 μm (±45 μm)),so that the cells can be visualized readily by a detection system withina narrow focal plane. If a dynamic focusing system is employed, it iscontemplated that flatness tolerances greater than 100 μm can betolerated. Accordingly, it can be preferable to use a support systemthat maintains the membrane and any captured cells in a substantiallyplanar orientation and within a suitably tight flatness tolerance topermit reliable detection. Depending on the detection system andrequirements post detection, the support system may be adapted topresent and/or maintain planarity of the membrane when dry and/or whenwet or moist after cells have been captured on the solid support afterpassing a cell containing solution through the solid support via poresdisposed within the solid support.

Various approaches described below allow the porous membrane 240 to beheld substantially flat after cells from a sample fluid are capturedthereon and other approaches may be apparent to those skilled in the artbased on the discussion herein.

In one embodiment, when the porous membrane 240 is wetted, surfacetension between the membrane 240 and membrane support 285 conforms thebottom surface of the membrane 240 to an upper mating surface of thesupport 285. For example, in one embodiment, the membrane support 285may be a fluid permeable, solid, substantially planar element that keepsthe membrane 240 in a substantially planar configuration, for example,when the membrane 240 is wetted. The support 285 is porous, and theupper mating surface is substantially flat and smooth. In anotherembodiment, the support 285 is coated with a non-toxic adhesive, forexample, polyisobutylene, polybutenes, butyl rubber, styrene blockcopolymers, silicone rubbers, acrylic copolymers, or some combinationthereof. When a downward pressure is applied, for example, from avacuum, the porous membrane 240 becomes loosely adhered to the support285, which results in the porous membrane 240 conforming to the surfaceof the support 285. The support member 285 is porous, and the uppermating surface is substantially flat and smooth. For example, in oneembodiment, the surface has a flatness tolerance of up to about 100 μm.

The diameter of the support 285 is approximately the same as the portionof the membrane 240 for receiving cells, and preferably the support 285has a substantially uniform thickness. The support 285 can have athickness in a range selected from the group consisting of approximatelyfrom 0.1 mm to 10 mm, from 0.5 mm to 5 mm, and from 1 mm to 3 mm.Materials suitable for making the porous support member 285 includeplastic, polycarbonate, high density polyethylene (HDPE), glass, andmetal. In one embodiment, the support member 285 is fabricated bysintering plastic particles made from poly (methyl methacrylate) havinga mean diameter of 0.15-0.2 mm held at a temperature near the meltingpoint of the particles and at a pressure sufficient to cause sinteringof the particles to fuse them together and form a uniform structure.

Although the membrane 240 and the support 285 are depicted as circular,this is illustrative only. In other embodiments, the membrane 240 and/orthe support 285 may be shaped as a square, a rectangle, an oval, etc. Ingeneral, the shape and the surface area of the support 285, if it isused, is selected such that the surface of the support 285 isapproximately the same size as or slightly smaller than the portion ofthe membrane 240 for receiving cells disposed thereon.

The membrane 240 is disposed in contact with the substantially flat,smooth surface of the support member 285 before the sample fluid ispoured onto the membrane 240. The generally flat surface helps keep themembrane 240 substantially flat after the sample fluid is drained. Thesupport 285 can also serve as a reservoir for fluid passed through themembrane 240, to provide the additional benefit of preventing themembrane 240 and viable cells disposed thereon from drying out duringthe detection process. Drying can be detrimental to the viability of thecells retained on the membrane 240.

The base 110 also includes a register feature 295 to ensure proper andconsistent positioning of the base 110 in a cell detection system. Inone exemplary embodiment, register feature 295 is an indentation on anouter surface of the base 110. When the base 110 is inserted into acorresponding structure having a mating feature, e.g., a spring loadedball bearing, a user will know the base is properly positioned when thebase 110 “locks” into place, or some other feedback is provided to theuser. Other registration techniques may be used, including thosedescribed above with respect to the interface between the ring 235 andthe wall 265.

In another embodiment, a cell capture system 500 (as depicted in FIGS.5A-5F) is largely similar to the cell capture system 100, includinghaving similar components, such as a cup 505, a base 510, and a lid 515.The cell capture system 500 also includes a membrane 540 disposed on thebottom of the cup 505, which is designed to interface with a supportmember 585 to help maintain planarity of the membrane 540 (see, FIG.5E). As the components of the cell capture system 500 share much incommon with those of the cell capture system 100 (indicated through theuse of common numbering), and have the same or similar properties asdescribed above with respect to the cell capture system 100, onlydifferences in various aspects of the components are described below.

As discussed with respect to cell capture system 100, protrusions 260and the notches 270 are also dimensioned to provide sufficientfrictional interfit so as maintain engagement when an appropriate torqueis applied to ring 235/base 110 relative to the upper portion of the cup230 at which point ring 235 breaks away from the upper portion of thecup 230 along parting plane 250. However, if the frictional interfitbetween ring 235 and base 110 is too great, it can be difficult tosubsequently remove ring 235 from base 110 for subsequent analysis. Theuse of latches attached to the cup which interfit with the base 110(see, FIGS. 5-7) can solve this problem. The latches permit theappropriate level of frictional interfit to break ring 235 from upperportion of the cup 230 but without requiring such friction to make itdifficult for a user to separate ring 235 from base 110. These featuresare discussed in more detail with reference to FIGS. 5-7.

Cup 505, depicted separately in FIGS. 5A, 5B, and 6A-6F, includes aplurality of latches 592 extending downward from an outer edge of thecup 505. The latches 592 are configured to engage correspondingprotrusions 594 on the base 510 (see, FIGS. 7A and 7C) While two latches592 are depicted, as few as one may be used, with an upper limitdictated solely by the size of the latches and the circumference of thesurface on which the latches 592 are mounted. The latches 592 may beresilient, such that when the cup 505 is initially engaged with the base510, the latches 592 flex around the ledges 594 before reengaging alower portion of the ledges 594. As the latches 592 engage lowerportions of the ledges 594, but not their sides, the latches 592restrict longitudinal separation of the cup 505 from the base 510, butdo not limit or increase the torque requirements to separate the cup 505from the ring 535. As with the ring 135, the ring 535 has a differentlydimension protrusion 660′ in comparison to the other protrusions 660 tofunction as a registration feature. The protrusion 660′ can berelatively long and extend into a space in the base 510 to allow foreasier grasping (e.g., with forceps) during removal of the ring 535.This space for engaging the protrusion 660′ may be even greater inembodiments where the base 510 has a flat surface 796 adjacent acircumferential opening 775, as depicted in FIGS. 7A and 7B. Grips 552are shown on the outer surfaces of cup 505 and base 510 (see, FIG. 5F),which can help a user create the appropriate force or torque withoutslippage to separate ring 535 from the upper portion of cup 505.

The arrangement with the latches 592 can help increase the stability ofthe cell capture system 500 during transport, and limits the chances thecup 505 becomes disconnected from the base 510 at any time beforedesired use. Given the extra stability provided by the latches 592, thering 535 may not be required to fit as snugly within the base 510 as inother embodiments. This can reduce the force required to remove the ring535 from the base 510 after cell capture, reducing the likelihood thering 535 becomes damaged or the membrane distorted, for example, outsidethe required flatness tolerance, during removal. Further, the additionof a longer protrusion 660′ and in a wider space may aid a user byrequiring less force to remove ring 535 from base 510.

In FIGS. 7A and 7C, base 510 is shown without a fluid permeable membranesupport. In FIG. 5E, the permeable member 585 is shown in the cellcapture system disposed underneath membrane 540.

In certain embodiments, the cell capture system, in particular theporous membrane 240, has a sterility assurance level less than 10⁻⁶,10⁻⁷, 10⁻⁸, or 10⁻⁹. This can be achieved, for example, by sterilizingthe cell capture system 100 or 500, via techniques known in the art, forexample, via autoclaving, exposure to ionizing radiation, for example,gamma radiation or exposure to a sterilizing fluid or gas, for example,ethylene oxide or vaporized hydrogen peroxide. The cell capture system100 can be enclosed within a receptacle (e.g., a bag), prior to, during,or after sterilization. The cell capture system 100 can be placed withina receptacle (e.g., a bag) and sealed (e.g., hermetically sealed) beforeterminal sterilization (e.g., via exposure to ionizing radiation).

(II) Cell Capture System Manufacture and Assembly

The cell capture systems 100 and 500, and their various components, canbe made and assembled using known techniques, including injectionmolding and machining. While the techniques are described below withrespect to the cell capture system 100, the same techniques can be usedfor the cell capture system 500 and its components. The cup 105 and thebase 110 may be made out of any substantially rigid material capable ofbeing sterilized. For example, the cup can be fabricated from materialsknown in the art, for example, plastics and metals, which may varydepending upon a number of factors, for example, ease of manufacture,cost, and the coupling system employed. For example, when the upperportion 230 and the ring 235 are integral components of the cupseparated by a frangible connection, the upper portion and ring can bemolded, for example, injection molded, using a polymer material, e.g.,polyethylene or polypropylene.

In one approach, the cup 110 with the upper portion 230 and the ring 235can be manufactured as one or more parts. Then membrane 240 is securedto the ring 235 about a periphery thereof, prior to positioning the cup105 within the base 110. It is understood, however, that depending uponthe embodiment and securing mechanism, the membrane may be attached tothe periphery of the ring prior to the ring 235 being attached to theupper portion 230 of the cup. Thereafter, the resulting ring andmembrane assembly can then be attached to the upper portion of the cupvia a connection such as a threadable connection, a bayonet connectionor an interference fit connection.

The various components can be a wide variety of sizes while still beingwithin the scope of the invention. In certain embodiments, the cellcapture system 100 is between about 1 inch and 5 inches tall (e.g.,approximately 3 inches), with a diameter of between about 1 inch and 3inches (e.g., approximately 2.25 inches). The cell capture system 100may be sized to hold from about 100 mL to about 200 mL of the fluidsample, although different sizes may be used to hold more or less fluid.

As described above, a registration feature between the ring 235 and thebase 110 may be used, so the step of positioning the cup 105 in the base110 puts the cup 105 in a predetermined circumferential orientationrelative to the base 110. If the support 285 is included, it should beplaced in the recess 280 prior to positioning the cup 105 in the base110.

(III) Cell Capture Method

As described above, FIGS. 1A-1F depict the components of an exemplarycell capture system 100, and FIGS. 5A-5F depict similar components of anexemplary cell capture system 500. The description below with respect tothe method of using the cell capture system 100 is also applicable tothe cell capture system 500. The cup 105 is coupled to the base 110through the interface between the ring 235 and the base 110. The lid 115may be provided on top of the cup 105 to protect the interior of the cup105 from being contaminated.

During use, in order to capture/harvest cells, a fluid sample is applied(e.g., poured) into the cup 105. Due to the tapers of the upper portion105, the fluid wets and passes through the membrane 240. The fluidtypically passes through the membrane assembly (e.g., through themembrane 240, and the support 285, if one is used) toward the base 110.Negative pressure, for example, a vacuum, can be used to draw fluidthrough the membrane 240 to the openings 290, and to help keep themembrane 240 substantially flat. The fluid application step can occurbefore, at the same time, or after application of the vacuum. It iscontemplated that the substantially non-autofluorescent membrane 240permits a flow rate therethrough of at least 5 or at least 10 mL/cm²/minwith a vacuum of about 5 Torr or about 10 Torr.

After the fluid is drawn through the cell capture system 100, anyparticles and/or cells in the fluid that cannot pass through themembrane 240 are retained on the upper exposed surface of the membrane240. After pouring the fluid into the cup assembly 100, the upperportion 230 may be separated from the ring 235, with the resultingassembly depicted in FIGS. 4A-4F (with an optional lid 405 and a plug410).

To separate the upper portion 230 from the ring 235, the user may applya sufficient force to break the connection. When the upper portion 230and the ring 235 are integrally formed, as in the exemplary embodiment,the user may twist the cup 105 relative to the base 110. Based on theoverlap between the protrusions 260 and the notches 270 providingresistance to such rotation, the upper portion 230 will separate fromthe ring 235 along parting plane 250. The force required depends on anumber of factors, including the thickness of the frangible connection245, and the cell capture system 100 is typically designed to require atleast 5-20 inch pounds (0.56-2.26 newton meters). The lid 405 may beplaced on top of the base 110 to protect the moistened membrane 240 andsupport 285 from contamination when the base 110 is transferred to adetection system, or when the base 110 containing the membrane 240 isincubated, for example, for 15 minutes to 8 hours to permit the capturedviable cells to proliferate. The plug 410 can be secured to a bottom ofthe base 110, for example, via finger protrusion 295, to prevent anyleakage of residual fluid, particularly when the base 110 is insertedinto other equipment, for example, into a stage that is itselfintroduced into the detection system.

In another embodiment, the rings 235, 535 can be placed onto an adapter800 (see FIGS. 8A-8E) following removal from the respective cell capturesystem 100 or 500. The adapter 800 can have similar notches 270 as thebases 110, 510, allowing for the rings 235, 535 to be held securely.Many different patterns for the notches 270 can be used, including thosedepicted in the bases 110, 510, though any complementary pattern to theprotrusions 260, 660 on the rings 235, 535 may be used. As describedabove, a unique notch 270′ may be used to uniquely orient the rings 235,535 when disposed on the adapter 800. A registration feature 895 (e.g.,a protrusion) can be located the adaptor 800 to orient the adaptor 800when placed in the scanning system (e.g., mated with a stage with amatching pattern to ensure proper orientation). As the rings 235, 535can be uniquely oriented within the adapter 800, and the adapter 800 canbe uniquely oriented within the scanning system, the membranes 240, 540can have a consistent and known orientation during scanning. Further, araised portion 885 may function similarly as the support members 285,585, namely by providing a surface on which a lower surface (i.e., theside without the cells) of the membranes 240, 540 may interface tomaintain planarity of an upper surface (i.e., the side with the cells)of the membranes 240, 540 during scanning.

Similar to the lid 405 (depicted in phantom in FIG. 4A), a lens 905(depicted in FIGS. 9A-9E) may be used with the adaptor 800 to cover thecontents therein (e.g., the membranes 240, 540) to preventcontamination. In some embodiments, the lens 905 may be removed duringscanning, while in other embodiments the lens 905 may remain on theadaptor 800 during scanning. A lower surface of the lens 905 may haveone or more features for mating with the adaptor 800, such as thedepicted wall in FIGS. 9A-9C. The wall can rest in a space between aninside wall and an outside wall of the adaptor 800, helping ensurestability of the lens 900 and full coverage of the membranes 240, 540during scanning. An upper surface of the lens 900 may be substantiallyflat or arcuate, and can be controlled to ensure proper scanningresults.

The cells can be stained at any point after capture with a viabilitystain or a viability staining system, for example, as discussed in U.S.patent application Ser. No. 13/875,969, so that it is possible toselectively detect and distinguish viable cells from non-viable cells.The cells may optionally be washed with a physiologically acceptablesalt and/or buffer solution to remove residual non-specifically boundfluorescent dye and/or quencher.

Once the cell capture system has been used to capture cells originallypresent in the fluid sample, and the cells stained as appropriate, theresulting membrane (still attached to the ring) can be inserted into astage (see, U.S. patent application Ser. No. 13/875,969) for insertioninto a suitable detection system. Exemplary detection systems aredescribed, for example, in International Patent Application No.PCT/IB2010/054965, filed Nov. 3, 2010, U.S. patent application Ser. No.13/034,402, filed Feb. 24, 2011, International Patent Application No.PCT/IB2010/054966, filed Nov. 3, 2010, U.S. patent application Ser. No.13/034,380, filed Feb. 24, 2011, International Patent Application No.PCT/IB2010/054967, filed Nov. 3, 2010, and U.S. patent application Ser.No. 13/034,515, filed Feb. 24, 2011. Other patent applications directedto such systems include U.S. Patent Publication Nos. US2013/0316394,US2013/0309686, US2013/0323745, and US2013/0316363.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein or attached hereto in the appendix isincorporated by reference for all purposes. The entire description ofU.S. Provisional Patent Application Ser. Nos. 61/641,805; 61/641,809;61/641,812; 61/784,759; 61/784,789; and 61/784,807 and U.S. patentapplication Ser. Nos. 13/875,914; 13/875,936, 13/875,969 and 13/886,004are incorporated by reference herein for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Various structuralelements of the different embodiments and various disclosed method stepsmay be utilized in various combinations and permutations, and all suchvariants are to be considered forms of the invention. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A cell capture system for receiving a fluidsample, the system comprising: (a) a cup comprising: (i) an upperportion, (ii) a ring having a periphery, wherein the upper portion isseparably coupled to the ring by a frangible connection comprising acircumferential groove that defines a parting plane between the upperportion and the ring so that the upper portion can be separated from thering upon manual application by a user of a rotational force sufficientto break the frangible connection, and (iii) a fluid permeable membraneattached to the periphery to produce a fluidic seal between the membraneand the ring, wherein a portion of the membrane is adapted to retaincells thereon; and (b) a base configured to receive the ring.
 2. Thesystem of claim 1, wherein the membrane portion (i) defines a pluralityof pores having an average diameter less than about 1 μm so as to permitfluid to traverse the portion of the membrane while retaining cellsthereon and (ii) is substantially non-autofluorescent when exposed tolight having a wavelength in a range from about 350 nm to about 1000 nm.3. The system of claim 1, wherein the membrane portion has a flatnesstolerance of up to about 100 μm.
 4. The system of claim 1, wherein thecup is adapted to direct a fluid, when introduced into the upperportion, toward the membrane portion.
 5. The system of claim 1, whereinthe ring is integrally formed with the upper portion.
 6. The system ofclaim 1, wherein the frangible connection comprises a thin wall at anintersection of the upper portion and the ring.
 7. The system of claim1, wherein the ring comprises a circumferential registration feature. 8.The system of claim 1, wherein the membrane is at least one of adhered,bonded, heat welded, and ultrasonically welded to the ring.
 9. Thesystem of claim 1, wherein the base defines a recess adapted to receivea membrane support.
 10. The system of claim 9, wherein the recessdefines a plurality of openings adapted to permit the passage of fluidtherethrough.
 11. The system of claim 1, wherein the base comprises aregistration feature.
 12. The system of claim 11, wherein theregistration feature comprises a depression defined by a surface of thebase.
 13. The system of claim 1, wherein the cup further comprises atleast one latch adapted to couple the cup to the base.
 14. The system ofclaim 13, wherein the at least one latch is adapted to resist separationof the cup and the base in a plane perpendicular to a parting plane. 15.A method of harvesting cells if present in a fluid sample, the methodcomprising: (a) introducing the fluid sample to the upper portion of thecup of claim 1; and (b) permitting the fluid to pass through themembrane portion.
 16. The method of claim 15, further comprising, afterapplying the fluid, separating the upper portion from the ring.
 17. Themethod of claim 16, wherein separating the upper portion from the ringcomprises applying a force sufficient to decouple the ring from theupper portion.
 18. The method of claim 17, wherein applying the forcecomprises twisting the cup relative to the base.
 19. A method ofmanufacturing a cell capture system of claim 1, the method comprisingthe steps of: (a) providing a ring having a periphery; (b) securing afluid permeable member to a periphery to produce a fluidic seal betweenthe membrane and the ring; and (c) positioning the ring having themembrane secured thereto within a base configured to receive the ring.20. The method of claim 19, wherein, prior to step (b), the ring isseparably coupled to the upper portion.
 21. The method of claim 19,wherein the positioning step comprises mating the cup with the base in apredetermined circumferential orientation.
 22. The method of claim 19,further comprising, prior to positioning the cup within the base,placing a porous support in a recess formed in the base.